Process for preparing chloramphenicol analogues

ABSTRACT

The present invention relates to a process for preparing analogues of chloramphenicol, characterized by culturing a microorganism capable of producing D-threo-2-acetamido-1-pnitrophenyl-1,3-propanediol (hereinafter designated as Substance C), and the derivatives of D-threo-2-propionamido-1-pnitrophenyl-1,3-propanediol (hereinafter designated as Substance A), D-threo-2-isobutyramido-1-p-nitrophenyl-1,3-propanediol (hereinafter designated as Substance B) and Substance C of which 3-hydroxy group is substituted by O-acetyl group, under aerobic conditions in a culture medium containing assimilable carbon sources to form and accumulate said analogues of chloramphenicol therein and isolating and recovering the same therefrom, said microorganism belongs to the genera Corynebacterium. These substances A, B and C are the derivatives of D-threo-2amino-1-p-nitrophenyl-1,3-propanediol of which 2-amino group is substituted by propionamide, isobutyramide and acetamide respectively.

United States Patent [191 Suzuki et al.

Sept. 3, 1974 PROCESS FOR PREPARING CHLORAMPHENICOL ANALOGUES [75] Inventors: Takeo Suzuki; Fusao Tomita; Haruo Honda; Kunikatsu Shirahata; Takashi Deguchi, all of Tokyo; Fumio Kato, Shizuoka, all of Japan [73] Assignee: Kyowa Hakko Kogyo Kabushiki Kaisha, Tokyo-to, Japan [22] Filed: May 21, 1973 [21] Appl. No.: 362,625

Related [1.8. Application Data [63] Continuation-impart of Ser. No. 137,696, April 26,

1971, abandoned.

[52] US. Cl. 195/96, 195/80 R [51] Int. Cl Cl2d 13/00 [58] Field of Search 195/80, 81, 82, 96

[56] References Cited UNITED STATES PATENTS 2,483,892 10/1949 Ehrlich et al. 195/80 R 3,751,339 7/1973 Suzuki et al. 195/96 Primary Examiner-Lionel M. Shapir9 [5 7] ABSTRACT The present invention relates to a process for preparing analogues of chloramphenicol, characterized by culturing a microorganism capable of producing D- threo-2-acetamido-1-p-nitrophenyl-1 ,3-propanediol (hereinafter designated as Substance C), and the derivatives of D-threo-2-propionamido-l-p-nitrophenyl- 1,3-propanediol (hereinafter designated as Substance A), D-threo-2-isobutyramidol -p-nitrophenyl-l 3- propanediol (hereinafter designated as Substance B) and Substance C of which 3-hydroxy group is substituted by O-acetyl group, under aerobic conditions in a culture medium containing assimilable carbon sources to form and accumulate said analogues of chloramphenicol therein and isolating and recovering the same therefrom, said microorganism belongs to the genera Corynebacterium.

These substances A, B and C are the derivatives of D-threo-2-aminol -p-nitrophenyl-l ,3-propanediol of which 2-amino group is substituted by propionamide, isobutyramide and acetamide respectively.

7 Claims, No Drawings PROCESS FOR PREPARING CHLORAMPHENICOL ANALOGUES RELATED APPLICATION The present application is a continuation-in-part of Ser. No. 137,696 now abandoned, filed Apr. 26, 1971 in the name of the present inventors.

The process of the present invention is directed to the preparation of chloramphenicol-related compounds by fermentation using inexpensive hydrocarbons or carbohydrates as raw materials. Such compounds have known utility as antibacterial medicine. Conventionally, analogues of chloramphenicol have been derived chemically from chloramphenicol obtained by the fermentation of a microorganism belonging to Actinomycetes or by chemical synthesis. A process for preparing analogues of chloramphenicol directly by fermentation using bacteria has never been reported in the art.

It has now been discovered that the above-mentioned types of chloramphenicol analogues can be prepared with a good yield by means of culturing a microorganent invention has the following advantages:

a. shorter period of culturing time, b. larger yields of the products, and

c. wider variety of suitable carbon sources (not only carbohydrates but also cheaper hydrocarbons such as n-paraffins, alcohols, organic acids etc.)

Various microorganisms which are classified into the genus Corynebacterium and other microorganisms closely related thereto may be used for the practice of the present invention. However, it is preferred to use, for example Corynebacterium hydrocarboclastus ATCC 15592 and Corynebacterium hydrocarboclastus ATCC 21628.

The above microorganism is freely available to the public and is on'deposit with the American Type Culture Collection.

It is preferred to use as carbon sources various hydrocarbon fractions containing n-paraffins, especially C to C preferably C to C n-paraffins. But it is also possible to use other carbon sources such as carbohydrates, organic acids, alcohols, etc. (e.g. glucose, sorbitol, sucrose, glycerol, acetic acid etc.) as far as they are assimilable by the microorganisms employed.

Various inorganic and organic nitrogen sources can be used as nitrogen sources for the present invention.

In carrying out the cultivation, hydrocarbons or carbohydrates are used as main carbon sources. They are supplemented, for example, with organic nitrogen sources such as com steep liquor, yeastextract etc., inorganic metallic salts such as iron, manganese, magnesium, potassium, sodium etc. and growth-promoting factors to provide a culture medium. The medium is sterilized and inoculated with the microorganism. The cultivation is carried out under aerobic conditions at -45C. During the cultivation, the pH is adjusted to 4-10, preferably 6-8, for example, by addition of urea solution, aqueous ammonia, ammonia or ammonium carbonate solution. The cultivation is completed after 2-7 days. The completion of the fermentation can be confirmed by the values of the chloramphenicol analogues obtained as determined by means of the paper disc method so as to reach a maximum.

The following non-limitative examples illustrate the present invention.

EXAMPLE 1 Corynebacterium hydrocarboclastus KY4339 (ATCC 21628) was cultured with shaking for 24 hours in a medium having a composition of 1.0 percent of meat extract, 1.0 percent of peptone, 0.5 percent of NaCl and 2 percent of sorbitol, and having a pH of 7.2 (before sterilization). The resultant seed culture was inoculated into 3.0 liters of a culture medium having the following composition in a 5-liter jar fermenter at a ratio of 10 percent by volume of seed to culture medium.

KH,PO Na HPQ 0.291 MgSO '7H O 0. 1% MnSO -4H O 0.00271 FeSO,-7H O 0.02% ZnSO ,-7H,O 0.00 W: (NH SO 0.5% CuCl -2H,O 0.00037! Corn steep 0.5% Yeast extract 0.5% liquor n-paraffin 10% (v/v) (mixture of C C The cultivation was carried out at 30C for hours with-agitation (650 rpm.) and aeration (1 liter/min) with sterilized air. The pH of the medium was automatically adjusted to 6.5 6.8 with aqueous ammonia. Each 100. ml of n-paraffin was supplemented to the medium three times after every 12 hours. The n-paraffin substrate was almost consumed after the completion of the fermentation.

The fermented liquor obtained 2 liters) by the removal of microbial cells from the broth was adjusted to a pH of 4.0. After addition of an equal amount of ethyl acetate, the fermented liquor was shaken for 24 hours to extract analogues of chloramphenicol in the ethyl acetate layer. The thus-extracted liquor (2.3 liters) was dehydrated with anhydrous sodium sulphate and evaporated at 35C under reduced pressure. The resultant residue was further extracted with ethyl acetate (100 ml). After centrifugation, the solvent layer was fed through a silica gel column (diameter: 4 cm; height: 20 cm) which was then washed thoroughly with chloroform. After this, a mixed solution of chloroform and methanol (:5) was fed through the column.

In thecourse of the elution Substance B was eluted into the fraction of from 200 ml to 500 ml. Substances A and C were respectively eluted into the fractions of from 600 ml to 1,000 ml and 1,200 to 1,700 ml. Each fraction was then separately collected and concentrated. Each concentrated fraction was dissolved in ethanol, and Substances A, B and C were then sepa- The isolated crystals of Substances A, B and C had lower antibiotic activities in vitro than that of the chloramphenicol, but had the same tendencies as to antibiotic spectrum. These substances had a maximum ultraviolet absorption at 273p. which was the same as that of chloramphenicol.

From the results obtained by elemental analysis, infrared spectrum, nuclear magnetic resonance specsubstance was eluted in the chloroform eluate. Further, Substances A, B and C were eluted in the eluate of a mixture of chloroform and methanol and were isolated as crystals in amounts of 4.2 g, 1.2 g and 3.4 g, respectrum, mass spectrometry, etc., it was found that Sub- 5 y The POW-mentioned unknown Substance stances A, B and C are respectively identical with the g) eluted in chloroform eluilte f crystalllled substances having the following structures, which have after elfapol'atlon ofchlomfomli and 1 Substance Was already been d iv d h i ll f hl h i determined as a mixture of acetyl derivatives of Sub- COL stances A, B and C by means of infra-red spectrum, nu-

clear magnetic resonance, elementaryanalysis, etc. 7 W g g M Concurrently, it was confirmed that Substances A, B D threo-2-propionamido-l-p- P Y J- and C were produced by deacylating these acetyl derivpropanediol (Substance A) ati 1 EXAMPLE 3 H I V g g The same strain as that used in Example 1 was cul- H NHCOCHZCH: tured for 70 hours using various carbon sources in a 1 t CH H similar manner to that described in Example 1. The re- 1 suIts obtained are shown in the following table. The H amounts of products formed are in grams per liter.

" TABLE Conc. Sub.B Sub.A Suh.C'. D g/l g/l g/l I g/l n-paraffin 10'7( 2.5 4.8 2.1 1.2 I m to 20) i sucrose 10% 1.5 4.2 2.2 1.3 glucose do. 0.7 2.3 1.0 0.5 sorhitol do. 0.9 1.5 0.8 0.4 ethanol 27: ata time 0.6 .l.2 0.6 0.2

(total: 10%) glycerol 10% 1.3 2.3 0.8 0.8 waste molasses do. 1.2 4.2 2.8 1.2 acetic acid 17: at a time 0.7 0.8 0.5 0.4

(total: 10%) Remarks:

Cone. Concentration of additives. vol /1 D mixture f acetyl dcrivatus nt'Suhslanccs A. B and C NHCOCE D-threo-Z-acetamido l -p-nitrophenyll ,3-

propanediol (Substance C) H NHC OCHa EXAMPLE 2 The same strain as that ,used in Example -1 was cultured in a similar manner to that described in Example 1 with the exception of that n-paraffin, namely, fraction of C to C was substituted by sucrose. After culturing for 70 hours, the fermented liquor obtained was treated in a similar manner to that described in Example 1. 1n this case, the concentrate obtained by extraction with ethyl acetate was fed through a silica gel column which was eluted with chloroform and a chloroform-methanol mixture. It was found that an unknown Having described the presentinvention, that which is sought to be protected is set forth in the following claims.

We claim:

1. A process for preparing D-threo-Z-acetamido-l-pnitrophenyl-l,3-propanediol and the derivatives of D- threo-2-propionamidol -p-nitrophenyll ,3- propanediol, D-threo-2-isobutyramidol -p-nitrophenyl-1,3-propanediol and said D-threo-2-acetamidol -pnitrophenyl-l,3-propanediol of which 3-hydroxy group is substituted by O-acetyl group by culturing a microorganism belonging to the genera Corynebacterium in a culture medium containing assimilable carbon sources under aerobic conditions so as to produce one of the above noted compounds in said medium, and recovering the same therefrom.

2. The process of claim 1 wherein said culturing occurs at 20C to 45C at a pH of 4 to 10.

3. The process of claim 1 wherein said carbon source is a C to C n-paraffin.

4. The process of claim 1 wherein said carbon source is a material containing sucrose or glucose as sugar source.

5. The process of claim 1 wherein 3-O-acetyl derivatives of D-threO-Z-propionamidol-p-nitrophenyl-l ,3- propanediol, D-threo-2-isobutyramidol -p-nitrophenyll ,3-propanediol and D-threo-2-acetamidol -pnitrophenyl- 1 ,3-propanediol are produced.

6. The process of claim 1 wherein the genera Corynebacterium is Corynebacterium hydrocarboclastus ATCC 15592.

7. The process of claim 1 wherein the genera Corynebargerium is Corynebacterium hydrocarboclastus ATCC 20 16.

UNITED STATES PATENT OFFICE CERTIFICATE OF CGRRECTION PATENTNO.: 3,833,476 r DATED eptember 3, 1974 r INVENTOR(S) Takeo Suzuki et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: Claim 7 should read as follows:

--7. The process of claim 1 wherein the genera Corynebacterium is Corynebacterium hydrocarboclastus ATC 21628.

Signed and Scaled this Fourth D3) 0f January 1977 [SEAL] Attest:

RUTH c. MASON c. MARSHALL DANN 8 11 Commissioner ufPaIents and Trademarks 

2. The process of claim 1 wherein said culturing occurs at 20*C to 45*C at a pH of 4 to
 10. 3. The process of claim 1 wherein said carbon source is a C10 to C20 n-paraffin.
 4. The process of claim 1 wherein said carbon source is a material containing sucrose or glucose as sugar source.
 5. The process of claim 1 wherein 3-O-acetyl derivatives of D-threo-2-propionamido-1-p-nitrophenyl-1,3-propanediol, D-threo-2-isobutyramido-1-p-nitrophenyl-1,3-propanediol and D-threo-2-acetamido-1-p-nitrophenyl-1,3-propanediol are produced.
 6. The process of claim 1 wherein the genera Corynebacterium is Corynebacterium hydrocarboclastus ATCC
 15592. 7. The process of claim 1 wherein the genera Corynebacterium is Corynebacterium hydrocarboclastus ATCC
 20016. 